In vivo protein-protein interaction mapping

Abstract

The present invention is directed to the use of fluorescence resonance envery transfer (FRET) to screen for protein-protein interactions. More specifically, this invention provides a method to screen for and identify proteins that interact with a protein of interest. The method involves using DNA encoding the protein of interest tagged with one member of a fluorescent protein pair, and DNA sequences encoding a library of proteins to be screened, in which the proteins are tagged to the other member of the fluorescent pair. The protein of interest is co-expressed in cells with the library of proteins to be screened, such that single cell expresses the protein of interest and a single representative of the tagged library. This population of cells is screened by flourescence resonance energy transfer. Cells where interations have occurred are selected, allowing identification of the protein that interacts with the protein of interest.

1 . A method to screen for and identify proteins that interact with a protein of interest comprising 
 (a) transforming an expression system, wherein the expression system comprises a plurality of cells that express a protein of interest tagged with one member of a fluorescent protein pair by a plurality of DNA molecules encoding a protein to be screened tagged with the other member of the fluorescent protein pair, wherein individual members of the plurality of cells are transformed by different DNA molecules encoding said protein to be screened;    (b) screening said transformed expression system by fluorescence resonance energy transfer (FRET) to determine whether an interaction has occurred; and    (c) identifying any cells within said transformed expression system where there has been a transfer of fluorescence between the fluorescent protein pair as indicating that an interaction between the protein of interest and a protein being screened has occurred.    
     
     
         2 . The method of  claim 1 , wherein the plurality of cells are mammalian cells or yeast cells.  
     
     
         3 . The method of  claim 2  wherein the plurality of cells are yeast cells.  
     
     
         4 . The method of  claim 1 , wherein the cells identified as having an interaction between the protein of interest and the protein being screened are isolated from the transformed expression system.  
     
     
         5 . The method of  claim 4 , wherein the protein being screened is isolated from the cell.  
     
     
         6 . The method of  claim 1 ,  2 ,  3 ,  4  or  5 , wherein the fluorescent protein pair is enhanced blue and green fluorescent proteins or enhanced cyan and yellow fluorescent proteins.  
     
     
         7 . The method of  claim 1 , wherein a second group of cells is isogeneic to the plurality of cells of the expression system except that said cells have a bacterial, microbial or viral pathogen and said second group is screened to identify proteins that interact with the protein of interest, wherein one further identifies whether different proteins interact with the proteins of interest in the pathogen containing cells as compared to the cells of the expression system.  
     
     
         8 . The method of  claim 1 , wherein a plurality of malignant cells also expresses the protein of interest and said cells are screened by the same method as used with the expression system to select and identify proteins that interact with the proteins of interest, in both groups and said proteins that interact with the protein of interest in said malignant cells are compared with the proteins that interact with the protein of interest in the plurality of cells of the expression system to identify whether different proteins interact in the malignant cell as compared to the plurality of cells of the expression system.